Method for discriminating red blood cells from white blood cells by using forward scattering from a laser in an automated hematology analyzer

ABSTRACT

A method for identifying, analyzing, and quantifying the cellular components of whole blood by means of an automated hematology analyzer and the detection of the light scattered, absorbed, and fluorescently emitted by each cell. More particularly, the aforementioned method involves identifying, analyzing, and quantifying the cellular components of whole blood by means of a light source having a wavelength ranging from about 400 nm to about 450 nm and multiple in-flow optical measurements and staining without the need for lysing red blood cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/044,636, filed on Feb. 16, 2016, issued as U.S. Pat. No. 9,638,621,which is a continuation of U.S. patent application Ser. No. 14/562,073,filed on Dec. 5, 2014, issued as U.S. Pat. No. 9,267,931, which is acontinuation of U.S. patent application Ser. No. 12/767,611, filed Apr.26, 2010, issued as U.S. Pat. No. 8,906,309, which claims priority toU.S. Provisional Application Ser. No. 61/172,999, filed Apr. 27, 2009,the disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Certain embodiments of this invention relate to a method foridentifying, analyzing, and quantifying the cellular components of wholeblood by means of an automated hematology analyzer and the detection ofthe light scattered and absorbed by each cell. More particularly, theaforementioned method may involve identifying, analyzing, andquantifying the cellular components of whole blood by means of multiplein-flow optical measurements using a single dilution of sample withoutthe need for lysing red blood cells.

2. Discussion of the Art

Some automated hematology analyzers are equipped with an optical benchthat can measure multiple in-flow optical measurements, such as lightscattering, extinction, and fluorescence, as described in U.S. Pat. Nos.5,631,165 and 5,939,326, both of which are incorporated herein byreference. Furthermore, U.S. Pat. Nos. 5,516,695 and 5,648,225, both ofwhich are incorporated herein by reference, describe a reagent suitablefor lysing red blood cells and staining nuclear DNA of membrane-lysederythroblasts to discriminate white blood cells from erythroblasts.Membrane-lysed erythroblasts are erythroblasts wherein the membranethereof has undergone lysis. U.S. Pat. No. 5,559,037, incorporatedherein by reference, describes the simultaneous detection oferythroblasts and white blood cell differential by means of a tripletriggering circuitry, which is used to eliminate noise signals from celldebris, such as, for example, membranes of lysed red blood cells, whichare located below the lymphocyte cluster along the Axial Light Loss(ALL) axis of a cytogram. However, the use of lysing agents to lyse redblood cells brings about certain difficulties and complications in thedetection of white blood cells. The lysing agent may be insufficientlystrong, thereby resulting in red blood cells being counted as whiteblood cells. Alternatively, the lysing agent may be excessively strong,thereby resulting in artificially low counts of white blood cells.Different samples require lysing agents of different strengths in orderto obtain accurate counts of white blood cells; accordingly, allhematology analyzers currently in use sometimes yield incorrect countsof white blood cells, listing various kinds of lysis-resistant red bloodcells as interfering substances.

In hematological assays aimed at determining parameters from human wholeblood, there are two physiological factors that present obstacles tosimple, rapid, and accurate determination of cell counts. One factor isthat, in typical fresh peripheral human whole blood, there are about1,000 red blood cells and about 50 platelets for each white blood cell.The other factor is that, while platelets are typically sufficientlysmaller than any other cell type to allow discrimination based on size,and most white blood cells are sufficiently larger than either red bloodcells or platelets to again allow discrimination based on size, two cellspecies in particular—red blood cells and lymphocytes, a subtype ofwhite blood cells—typically overlap in size distribution (as well as intheir scattering signatures) to a sufficient degree to makediscrimination based on size prone to gross error. Therefore, whendetermining red blood cells mainly by size discrimination, the asymmetryin concentration can work in one's favor, since the occasional whiteblood cell misclassified as a red blood cell will not, generally, affectthe overall accuracy of the measured concentration of red blood cells toany appreciable degree; however, the converse is not true, and anyunaccounted-for interference from red blood cells in determining theconcentration of lymphocytes (and, by extension, the overallconcentration of white blood cells) can yield very inaccurate results.

Consequently, methods have been developed in the prior art to handlethis large asymmetry and size overlap and still provide useful resultsin an acceptable time frame. One standard method employed in the priorart has been to separate the blood sample to be analyzed into at leasttwo aliquots, one destined for red blood cell and platelet analysis, andone for white blood cell analysis. The aliquot destined for white bloodcell analysis is mixed with a reagent solution containing a lysingreagent that attacks the membranes of the red blood cellspreferentially, or faster than it attacks those of the white bloodcells. Partially on account of their loss of hemoglobin through thecompromised membrane, and partially on account of their attendantreduction in size, the resulting lysed red blood cells becomedistinguishable from lymphocytes based on their respective scatteringsignatures. Another method employed in the prior art involves usingnucleic acid dyes to provide a fluorescent distinction between the redblood cells and the white blood cells. White blood cells contain anucleus containing DNA. When these white blood cells are identified viaa fluorescent label, they can be distinguished from mature red bloodcells, whose nuclei have been expelled in the maturation process.

Both of these methods have drawbacks. First of all, the lysing reagentused to dissolve the red blood cells can attack the white blood cells aswell, reducing their integrity and eventually dissolving them, too. Thisis particularly a problem with white blood cells that are alreadyfragile in the first place, due to some pathological condition (such,as, for example, chronic lymphocytic leukemia). At the other end aretypes of red blood cells (such as, for example, those found in neonates,and in patients with thalassemia, sickle-cell anemia, and liver disease)which are naturally resistant to lysis, and which therefore tend topersist as interferents in white blood cell assays involving lysis. Inorder to reduce the likelihood of either degradation of white bloodcells or interference from unlysed red blood cells (either of whichwould jeopardize the accuracy of the overall white blood cellconcentration measurement), a careful combination of concentration oflysing agent, temperature control, and incubation time can be used. Insome cases, the user is offered several test options with differentlysing conditions, thereby allowing the user to tailor the assay to thesubject patient sample. This tailoring, however, is a complex solution,which additionally either requires prior knowledge of the state of thepatient, or must be used as a reflex test following a standard completeblood count (CBC).

Regarding the fluorescence-based approach at discriminating between redblood cells and lymphocytes, the main obstacle is the measurement rate.When white blood cells are measured at the same time as red blood cellsand platelets, the presence of red blood cells sets an upper limit tothe concentration that can be sent through the analyzer withoutincurring in coincidences at an unacceptably high rate; the dilutionratio used to achieve such concentration, in turn, limits the rate atwhich white blood cell events are being counted; and in order to obtainthe counting precision expected of the analyzer, this relatively lowrate of white blood cell event acquisition, in turn, forces longacquisition times. For example, the concept of measuring all of thecomponents of blood from a single sample in one pass was disclosed inU.S. Pat. No. 6,524,858. As noted in that disclosure, the method wouldbe capable of a cycle time of 88 seconds, or about 41 CBC/hr. Thisthroughput is far lower than that achievable by most automatedhematology analyzers commercially available today, severely limiting itscommercial usefulness. The CELL-DYN® Sapphire® hematology analyzer, asanother example, presently offers a test selection (requiring yetanother aliquot of sample in addition to those used in the red bloodcell/platelet assay and in the white blood cell assay) employing anucleic-acid dye capable of differentiating between intact (unlysed) redblood cells and lymphocytes. This test selection uses the dye primarilyto differentiate between mature red blood cells and reticulocytes, asubset of immature red blood cells that retain dye-absorbing RNA in thecytoplasm. While it would technically be possible to count the whiteblood cells using this same assay, as they are sufficientlydifferentiated by fluorescence from either red blood cells orreticulocytes to obtain the desired accuracy, the relatively lowconcentration of white blood cells in the dilution used makes it animpractical option to achieve the required statistical precision. Such ascheme would require an acquisition time of approximately 75 seconds,limiting throughput to only 48 CBC/hr. Accordingly, although thisapproach is theoretically feasible, a much higher throughput would berequired in order for this approach to become practical commercially.

A single-dilution approach presents many potentially attractivebenefits. One of them is the elimination of multiple aliquots. Thisfeature drastically simplifies the fluidic architecture of the system,since it requires only a single container (instead of two or more) inwhich to mix the blood sample and the reagent solution, and only asingle system (such as, for example, a precision metering syringe andassociated driver motor and control electronics) for measuring anddelivering the reagent solution to the mixing container. It also affordsan attendant reduction in the number of valves, the number of valveactuators, the number of individual segments of tubing, and the numberand quantity of reagents necessary to implement the desired assay.Another benefit is the elimination of the process of lysing red bloodcells. This feature reduces drastically the uncertainties associatedwith lysis-resistant red blood cells and with lysis-prone lymphocytes;it eliminates the need for the time-consuming and sensitive lysisincubation period; and, additionally, it eliminates a significantportion of the software dedicated to operate the analyzer, as previouslyseparate test selections are combined in a single procedure. Anotherbenefit accrues from the overall reduction in complexity of the analyzerdue to the individual changes just described.

There are additional potential attendant reductions in complexity.Hematology analyzers designed for high throughput may also generallyinclude additional transducers in addition to the flow cytometersubassembly incorporated therein, such as, for example, one or moreimpedance transducers to count, size, and identify some subpopulationsof blood cells, and a colorimetric transducer to determine thehemoglobin-related parameters of blood. A single-dilution approach couldeliminate the need for additional impedance transducers, for acolorimetric transducer, or for both impedance transducers andcolorimetric transducers, if the analyzer were capable of achievingsufficient speed, precision, and accuracy in measurement to render thesedeletions practical. Because the colorimetric transducer for bulkmeasurement of hemoglobin requires the use of a strong lysing agent todissolve the membranes of the red blood cells (the lysing agenttypically being in addition to the milder lysing agent used in the whiteblood cell assays), elimination of the colorimetric transducer may alsoeliminate the need for an additional on-board strong lysing agent inaddition to that used in the flow cytometer subassembly. The reductionin complexity, whether from (a) simply replacing the flow cytometersubassembly of the prior art with a single-dilution subassembly whilemaintaining a separate colorimetric transducer or an impedancetransducer or both, or from (b) additionally incorporating all thefunctions of impedance transducers and colorimetric transducers into asingle-dilution analyzer, may result in a substantial improvement in thereliability of the instrument, because the number of parts subject tofailure would be reduced, and because the number of componentsgenerating heat (which can reduce the lifetime of some components) wouldbe reduced. This potential improvement in reliability would likewiseprovide a major improvement in the instrument's service profile, withless maintenance required, fewer service calls required, and a lowercost for those calls that do occur, on account of the increasedserviceability of a simplified instrument architecture, i.e., aninstrument having fewer components. Beside the reliability improvements,a simplification in the instrument architecture would also reduce itscost, on account of both a reduced part count and simplified assemblyand testing activities during its manufacture.

All of these benefits, however, are overshadowed in the prior art by thelow throughput of the disclosed method. In other words, thesingle-dilution feature disclosed in the prior art is only one of theenabling elements of a superior analyzer. It would be desirable toenhance the single-dilution approach with a high measurement rate inorder to also provide the throughput performance commonly expected ofcommercial hematology analyzers, and typically expected of analyzersdesigned for high-volume environments.

Therefore, it would be desirable to develop a method for identifying,analyzing, and quantifying the cellular components of whole blood bymeans of multiple in-flow optical measurements without the need forlysing red blood cells.

SUMMARY OF THE INVENTION

A method and a hematology analyzer for performing the method areprovided. In certain embodiments, the hematology analyzer comprises: (a)a flow cell through which a whole blood sample is introduced at a highrate or at a low rate; (b) a laser having a wavelength in the range offrom about 400 nm to about 450 nm for directing light to the flow cell;(c) a plurality of detectors for detecting the interaction of light withcells on a plurality of optical measurement channels; and (d) a dataanalysis workstation comprising: i. programming to differentiate andcount white blood cells in the sample using multiple in-flow opticalmeasurements obtained from a first portion of the sample that has beenintroduced into the flow cell at a high rate; ii. programming todifferentiate and count red blood cells and platelets in the sampleusing multiple in-flow optical measurements obtained from a secondportion of the sample that has been introduced into the flow cell at alow rate. In certain cases the workstation may further comprise: iii.programming to measure hemoglobin-related parameters on a cell-by-cellbasis, as discussed in greater detail below.

In one aspect, the method comprises the steps of: (a) providing anautomated hematology analyzer capable of measuring light extinction,light scattering, and fluorescence, the automated hematology analyzerbeing equipped with a laser having a wavelength in the range of fromabout 400 nm to about 450 nm; (b) providing a diluent for diluting asample of blood which, in certain cases, contains a sphering agent; (c)providing a sample of whole blood; (d) mixing the diluent and the sampleof whole blood; (e) differentiating and counting white blood cells bymeans of multiple in-flow optical measurements with a high rate ofsample introduction; (f) differentiating and counting red blood cellsand platelets by means of multiple in-flow optical measurements with alow rate of sample introduction; and (g) measuring the concentration ofhemoglobin of the whole blood sample by measuring the concentration ofhemoglobin in each individual red blood cell by means of multiplein-flow optical measurements and using the red blood cell counts fromstep (f) to determine the concentration of red blood cells in the wholeblood sample.

In order to measure the concentration of erythroblast cells, the methodcomprises the additional steps of (a) staining erythroblast nuclei witha nuclear stain; (b) detecting erythroblasts by means of at least one ofextinction, light scattering, and fluorescence from the nuclear stain;and (c) differentiating and counting the erythroblast cells byalgorithms that analyze the separation of erythroblasts from other cellpopulations.

In order to measure the percentage of reticulocytes in the entirepopulation of red blood cells, the method comprises the additional stepsof (a) staining reticulocytes with an RNA stain; (b) detectingreticulocytes by means of at least one of extinction, light scattering,and fluorescence; and (c) differentiating reticulocytes and quantifyingthe reticulocyte percentage of red blood cells in the sample byalgorithms that analyze the distribution of fluorescent signals in thered blood cell population.

The method described herein can further include the steps of (a) storingdata for the analysis of the sample of whole blood, (b) reportingresults for the analysis of the sample of whole blood, and (c) analyzingthe sample of whole blood by at least one algorithm to count anddifferentiate white blood cells, erythrocytes, platelets, erythroblasts,and reticulocytes.

In one embodiment, the sample of blood is analyzed without any manualpreparation (save homogenization of the sample in the sample collectiontube by repeated inversions) in the Open Mode of the hematologyanalyzer. The samples prepared for onboard analysis using thediluent/sheath reagent are passed through the electro-opticalflow-cytometry system described herein, whereupon the electronic logicof the system and the algorithm(s) of the system differentiate each cellpopulation based on volume of the cells, refractive index of the cells,fluorescence intensity of the cells, presence and conformation of anynucleus inside the cells, presence and quantity of any cytoplasmicgranules inside the cells, and the location and pattern of each clusterof cells in cytograms constructed from combinations of two or more ofthe optical measurements, or in histograms constructed from projectionsof the optical measurements along a chosen axis. As would be understood,when taking measurements at a high rate of flow, not all of the cellsmay be in single file. At a high rate of flow, the white blood cells(WBCs) may appear to be strung on in single file with good separationbetween individual cells, and the red blood cells (RBCs) may befrequently coincident with the WBCs. At a high flow rate, the IAStrigger allows the analyzer to ignore the RBCs. At lower flow rates, theRBCs should generally be in single file so they can be counted. Theintermediate angle scattering (IAS) trigger qualifies signals from whiteblood cells. To be qualified as a valid white blood cell signal, i.e., asignal generated by a white blood cell, the amplitude of the signal mustbe above the intermediate angle scattering (IAS) trigger threshold; thealgorithm(s) of the system carry out the function of differentiating thesub-populations of the white blood cells by using the intensity of thesignals from a plurality of optical detectors, and the shape and thenumber of clusters.

The use of a laser having a wavelength ranging from about 400 nm toabout 450 nm aids in the separation of red blood cells from white bloodcells. The use of the aforementioned laser enables the hematologyanalyzer to function without the need for a lysing agent for red bloodcells in order obtain an accurate count of white blood cells.

In one embodiment, the analyzer uses one single dilution of whole bloodto count all of the cells in the sample. This count is achieved by usingtwo different sample introduction rates for the diluted blood into theflow cell. The white blood cell count uses a sufficiently high sampleintroduction rate and an intermediate angle scattering (IAS) trigger inorder to exclude platelets and red blood cells. The overall sampleintroduction rate for counting red blood cells and platelets is lowerthan the overall sample introduction rate used for counting white bloodcells. This technique allows a sufficient number of white blood cellevents to be collected in less than 40 seconds of counting time.Introduction of the diluted sample into the flow cell can be carried outby injection. The actual rates of sample introduction can be determinedby one of ordinary skill in the art without undue experimentation. Forexample, in certain embodiments a low rate of sample introduction may bein the range of about 1,000-50,000 cells per second (for samples withtypical concentrations found in normal adult blood); while in certainembodiments a high rate of sample introduction may be in the range ofabout 50,001-300,000 cells per second (for samples with typicalconcentrations found in normal adult blood).

In addition to the foregoing embodiments, the apparatus and method ofthis invention may employ elements representing a reduction in thenumber of corresponding elements conventionally used in currenthematology analyzers and flow cytometers. These elements are: (a) areagent system, free of lysing agent, that includes: i. a diluent andii. a RNA- and DNA-staining fluorescent dye, or a combination of twodifferent dyes that preferentially bind, respectively, to RNA and DNA;(b) a sample aspiration assembly capable of delivering a portion of asample; (c) a single container for holding such portion and for mixingof such portion with the reagent solution; (d) one or more subsystemsfor metering and delivery of the appropriate amount of reagents into thesample aliquot container; (e) a single subsystem for staging theresulting solution of sample aliquot and reagent to the optical flowcell; (f) fluidic components necessary for rinsing the sample path andfor waste disposal. In certain cases, the diluent can contain spheringagent, which, as described below, can be used in the detection of RBCs.As would be apparent, the diluent and the dye can be stored separatelyfrom one another and, in certain cases, can be combined with one anotherbefore use.

In one embodiment, the analyzer contains a colorimetric transducer forbulk detection and quantification of hemoglobin, appropriate fluidics,and appropriate electronics necessary to support the hemoglobin assayperformed on such a transducer. In another embodiment, the analyzer doesnot possess a separate colorimetric transducer for the bulk measurementof hemoglobin (thereby eliminating the supporting fluidics andsupporting electronics), having incorporated thehemoglobin-quantification function of such a transducer into thefunction of the flow cell in which is performed a single-dilution assayfree of a lysing agent.

Also provided is a method of identifying and counting reticulocytes andnucleated red blood cells in a sample of blood. In certain embodiments,this method comprises: (a) providing an automated hematology analyzerequipped with a laser having a wavelength in the range of from about 400nm to about 450 nm; (b) mixing a diluent comprising a fluorescent dye,e.g., from the SYTO family with a sample of whole blood, therebystaining reticulocytes and nucleated red blood cells in the sample withthe fluorescent dye; and (c) detecting the reticulocytes and nucleatedred blood cells in the sample by detecting fluorescence emitted by thefluorescent dye using the automated hematology analyzer. In certaincases, the blood sample can be introduced into the flow cell of theanalyzer at a high rate, and at a low rate, as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the illumination anddetection optics of an apparatus suitable for generating signals fromcells on multiple detection channels for differential analysis.

FIG. 2 is a flow chart illustrating the reduction in subsystems and thereduction in overall complexity attendant with the introduction of asingle-dilution analyzer that does not require a lysing agent.

FIG. 3 is a cytogram of unlysed whole blood, wherein the wavelength ofthe light source was 405 nm. It can be seen that there is clearseparation between platelets, red blood cells, and white blood cells.The number of white blood cells counted is too low, compared to theother populations, to allow a white blood cell differential to beperformed. In order to increase the proportion of white blood cellevents that are collected, an intermediate angle scattering (IAS)trigger threshold value corresponding to about 8000 on the IAS axis ofthe cytogram is required.

FIG. 4 is a cytogram of the white blood cells of an unlysed clinicalblood sample with a high rate of sample introduction (injection) and anintermediate angle scattering (IAS) trigger threshold at channel 7000.The white blood cell differential is shown, with monocytes in the areadesignated by the letter “M”, lymphocytes in the area designated by theletter “L”, neutrophils in the area designated by the letter “N”, andeosinophils in the area designated by the letter “E.” The classificationis achieved without a clustering algorithm.

FIGS. 5A, 5B, 5C, and 5D are cytograms of the prior art illustrating asample with lysis-resistant RBCs interfering with the WBC and lymphocytecounts, presenting a lymphocyte percentage of 95%. The cytograms weregenerated by a CELL-DYN® Sapphire® hematology analyzer using aconventional lysing agent of low lytic strength. The wavelength of thelaser, 488 nm, was not in the range described herein (i.e., 400 nm to450 nm).

FIGS. 6A, 6B, 6C, and 6D are cytograms illustrating the same sample asin FIGS. 5A, 5B, 5C, 5D and presenting a lymphocyte percentage of 48%.The cytograms were generated by a CELL-DYN® Sapphire® hematologyanalyzer using a conventional lysing agent of high lytic strength. Thewavelength of the laser, 488 nm, was not in the range described herein(i.e., 400 nm to 450 nm).

FIGS. 7A, 7B, 7C, and 7D are cytograms illustrating the same sample asin FIGS. 5A, 5B, 5C, and 5D and presenting a lymphocyte percentage of55%. The cytograms were generated by a CELL-DYN® Ruby® hematologyanalyzer using a conventional lysing agent of an intermediate lyticstrength. The wavelength of the laser, 633 nm, was not in the rangedescribed herein (i.e., 400 nm to 450 nm).

FIG. 8 is a cytogram illustrating the same sample as in FIGS. 5A, 5B,5C, and 5D and presenting a lymphocyte percentage of 12%. The cytogramswere generated by a prototype hematology analyzer using a 405-nm laserand the single-dilution techniques described herein without a lysingagent. Lymphocytes are in the area designated by the letter “L.”

DETAILED DESCRIPTION

As used herein, the expressions “axial light loss” and “ALL” refer tothe measurement of the total light lost from the laser beam when aparticle passes through the beam, and typically measured with a detectorsubtending a range of angles (measured from the cell or particle understudy) from 0° (the optical axis of the system) to about 1°. Thisparameter relates to measurement of light extinction, which compriseslight lost through scattering as well as absorption; in the absence ofabsorption, it is a measurement that correlates broadly with the size ofcells or particles passing through the optical detection system.

As used herein, the expressions “small angle scattering” and “SAS” referto the measurement of forward light scattering at small angles fromabout 1° to about 3°. For certain laser light wavelengths, e.g., for awavelength of 633 nm, this parameter relates to measurement of the sizeof cells. For certain other laser light wavelengths, e.g., for awavelength of 405 nm, this parameter relates to measurement of therefractive index of cells, which in turn is a measure of cytoplasmiccontent, such as, e.g., hemoglobin.

As used herein, the expressions “intermediate angle scattering” and“IAS” refer to the measurement of forward light scattering atintermediate angles from about 3° to about 10°. This parameter relatesbroadly to measurement of the complexity of a cell. As used herein, theterm “complexity” refers to the composition of a cell. Some cells havemitochondria, ribosomes, and a nucleus, while other cells lack one ormore of the foregoing components. The measured intensity of IAS dependsto some degree on the heterogeneity of the contents of a cell (orparticle) passing through the illumination beam of a cytometer. Theintensity of IAS signals can be thought of as a measure of thecomplexity of the contents of the cell, i.e., the presence oforganelles, such as, for example, nuclei, vacuoles, mitochondria, etc.For certain laser light wavelengths, the intensity of IAS signalsadditionally carries information on the average refractive index ofcells.

As used herein, the expressions “polarized side scattering” and “PSS”refer to polarized (i.e., polarization-maintaining) light scatteringinto a cone centered around the angle of 90° and having an angularhalf-width of between around 10° and around 75°. This parameter relatesbroadly to measurement of lobularity. The nuclei of cells have variousshapes that may result in one to five lobules, inclusive. Arepresentative example of a cell with multi-lobed nucleus is a segmentedneutrophil; higher lobularity tends to correlate with increased PSSsignals.

As used herein, the expressions “depolarized side scatter” and “DSS”refer to depolarized (i.e., polarization-rotating) light scattering intoa cone centered around the angle of 90° and having an angular half-widthof between around 10° and around 75°. This parameter relates broadly toa measurement of granularity. One main subclass of leukocytes isgranulocytes, a category of cells that includes neutrophils,eosinophils, and basophils, and is characterized by the presence ofcytoplasmic granules. A portion of the granules in eosinophils displaysa crystalline arrangement, leading to an increased DSS signal.

As used herein, the term “trigger” means the minimum electrical voltagethat an electrical signal must exceed to be considered valid.

As used herein, the term “erythroblast” means any of the nucleated cellsin bone marrow that develop into erythrocytes. As used herein, the term“erythrocyte” means the reddish, non-nucleated, disk-shaped blood cellthat contains hemoglobin and is responsible for the color of blood.

One or more detectors are preferably placed in the paths of lightemanating from the flow cell for measuring ALL, SAS, and IAS, or asubset of ALL, SAS, and IAS, or ALL and a detector measuring thecombined ranges of SAS and IAS; as well as PSS and DSS. ALL-measuringsubsystems collect light transmitted in the main beam of laserillumination, while scatter-measuring subsystems collect light outsidethe main beam. In ALL-measuring subsystems, the signal of interest is anegative signal subtracted from the steady-state laser signal, whereasin scatter-measuring subsystems (including SAS, IAS, PSS, and DSS), thesignal is a small positive signal imposed on a very low background lightlevel. IAS collection is similar to SAS collection, except the light isscattered at larger angles from the incident laser beam. In a preferredembodiment, ALL is collected by a detector having an angular half-widthabout 0.3° horizontally and about 1.2° vertically (measured from theoptical axis of the system), and IAS is collected by a detectorsubtending angles between about 2° and about 10° from the laser axis.

As used herein, the expression “Open Mode” means a method of presentingthe sample in an open tube to the automated instrument by a humanoperator. As used herein, the expression “Closed Mode” means a method ofpresenting the sample in a capped tube to the automated instrument by arobotic mechanism.

As used herein, the expression “measuring cells” refers to enumeratingcells by means of optical measurement techniques to determine, e.g.,size, refractive index, complexity, lobularity, granularity, andfluorescence when the cells are stained with a particular dye orfluorochrome.

The symbol “(s)” following the name of an object indicates that eitherthe object alone or a plurality of the objects is being referred to,depending upon the context of the statement surrounding the mention ofthe object or objects.

As used herein, the term “leukocyte” means white blood cell. Unlike redblood cells, white blood cells occur in many different types. Examplesof leukocytes include granulocytes (further subdivided into, e.g.,neutrophils, eosinophils, and basophils), lymphocytes, and monocytes.The expression “reference method” means a method of the prior artagainst which a test method is compared.

The term “sickle cell” means a red blood cell shaped like a sickle. Asickle cell is typically resistant to a lytic reagent.

The term “thalassemic” relates to a genetic blood disorder in which thebone marrow cannot form sufficient red cells and red cell survival isalso reduced.

The term “lymphocyte” means a white blood cell that matures in lymphnodes, the spleen, and other lymphoid tissues, enters the blood, andcirculates throughout the body.

The expressions “nucleated red blood cell” and “erythroblast” mean animmature red blood cell that still contains a nucleus.

As used herein, the term “noise” includes, but is not limited to, suchsubstances as lysed red blood cells in particulate form, cell debris,and platelet clumps.

As used herein, the term “event” means a particle generating a signalsufficient to trigger one or more detector, e.g., the IAS detector,whereby that detector signals the analyzer to collect measurements ofthat particle on all the detectors enabled on the analyzer, e.g., ALL,IAS, PSS, and DSS. Particles include, but are not limited to, are whiteblood cells (WBC), red blood cells (RBC), RBC fragments, platelets(PLT), lipids, and platelet clumps.

As used herein, the terms and phrases “diluent”, “sheath”, “sheathdiluent”, “diluent/sheath”, and the like, mean a reagent used as diluentand as sheath fluid of the type suitable for use with hematologyanalyzers such as the CELL-DYN® Sapphire®, CELL-DYN® Ruby®, CELL-DYN®3000 series, and CELL-DYN®4000 series hematology analyzers, whichreagents are commercially available from a variety of sources, includingAbbott Laboratories, Santa Clara, Calif.

In certain cases, a hematology analyzer may include a source of light, alens or system of lenses, a flow cell, and appropriate detectors, whichcomponents and functions thereof in a flow cytometry system arewell-known to those of ordinary skill in the art. See, for example, U.S.Pat. Nos. 5,017,497; 5,138,181; 5,350,695; 5,812,419; 5,939,326;6,579,685; 6,618,143; and U.S. Patent Publication Nos. 2003/0143117,US20080153170, US20080158561 and US20080268494, where exemplary sourcesof light, lenses, flow cells, and detectors are described in greaterdetail. All of the aforementioned references are incorporated herein byreference. Lasers, lenses, flow cells, and detectors suitable for use inthis invention are commercially available from a variety ofmanufacturers, including Abbott Laboratories, Santa Clara, Calif.

Certain embodiments of the method described herein involve an automatedmethod for simultaneous analysis of white blood cells, white blood celldifferential (which identifies the proportion of neutrophils,eosinophils, basophils, lymphocytes and monocytes in the sample, andoptionally of additional types of immature white blood cells, such as,e.g., immature granulocytes, blast cells, banded neutrophils, andvariant lymphocytes), erythroblasts, reticulocytes, red blood cells, andplatelets in liquid, such as, for example, blood. Other biologicalfluids, such as, for example, cerebrospinal fluid, ascites fluid,pleural fluid, peritoneal fluid, pericardial fluid, synovial fluid,dialysate fluid, and drainage fluid, can be used to determine variousparameters of these fluids.

The optical subassembly of an exemplary hematology analyzer isschematically illustrated in FIG. 1. One of ordinary skill in the artwould recognize that the choice, number and design of the components(e.g., the type of laser used, the number and specifications of theoptical components, etc.) can vary greatly between analyzers and, assuch, the hematology analyzer of FIG. 1 is provided as an example andshould not be used to limit this disclosure. For example, in certaincases a hematology analyzer may or may not detect fluorescence.

Referring now to FIG. 1, in one embodiment, an apparatus 10 comprises asource of light 12, a front mirror 14 and a rear mirror 16 for beambending, a beam expander module 18 containing a first cylindrical lens20 and a second cylindrical lens 22, a focusing lens 24, a fine beamadjuster 26, a flow cell 28, a forward scattering lens 30, a bulls-eyedetector 32, a first photomultiplier tube 34, a second photomultipliertube 36, and a third photomultiplier tube 38. The bullseye detector 32has an inner detector 32 a for ALL detection and an outer detector 32 bfor IAS detection.

In the discussion that follows, the source of light can be a laser.However, other sources of light can be used, such as, for example, lamps(e.g., mercury, xenon), light-emitting diodes (LEDs), andhigh-brightness LEDs. The light source can emit a beam of light at awavelength in the range of from about 400 nm to about 450 nm, e.g., inthe range of from about 400 nm to about 430 nm. For example, a laserthat emits light at a wavelength of about 405 nm or 413 nm can beemployed. In one embodiment, the source of light 12 can be a verticallypolarized 405-nm diode Cube laser, commercially available from Coherent,Inc., Santa Clara, Calif. Operating conditions for the laser can besubstantially similar to those of lasers currently used with automatedhematology analyzers.

Additional details relating to an exemplary flow cell, exemplary lenses,an exemplary focusing lens, an exemplary fine-beam adjust mechanism, andan exemplary laser focusing lens can be found in U.S. Pat. No.5,631,165, incorporated herein by reference, particularly at column 41,line 32 through column 43, line 11. The exemplary forward optical pathsystem shown in FIG. 1 includes a spherical plano-convex lens 30 and atwo-element photo-diode detector 32 located in the back focal plane ofthe lens. In this configuration, each point within the two-elementphotodiode detector 32 maps to a specific collection angle of light fromcells moving through the flow cell 28. The detector 32 can be abulls-eye detector capable of detecting ALL and IAS. U.S. Pat. No.5,631,165 describes various alternatives to this detector at column 43,lines 12-52.

In this example, the first photomultiplier tube 34 (PMT1) measures DSS.The second photomultiplier tube 36 (PMT2) measures PSS, and the thirdphotomultiplier tube 38 (PMT3) measures fluorescence in a range ofwavelengths that is consistent with the emission spectrum of afluorescent dye used, e.g., between around 450 nm and around 550 nm.Side-scattering and fluorescent emissions are directed to thesephotomultiplier tubes by dichroic beam splitter 40, which transmits andreflects efficiently at the required wavelengths to enable efficientdetection, and beam splitter 42 (optionally a polarizing beam splitter).U.S. Pat. No. 5,631,165 describes various additional details relating toexemplary photomultiplier tubes at column 43, line 53 though column 44,line 4.

In particular cases and as would be readily apparent, the filters usedin the fluorescence detection system (such as, e.g., filter 56 in frontof photomultiplier 38) can filter out the light produced by the laser,thereby permitting only fluorescent light to reach the photomultiplier.In one embodiment, a fluorescence filter suitable for use with a laserhaving a wavelength of 405 nm is used.

In certain cases, sensitivity can be enhanced at photomultiplier tubes34, 36, and 38 by using an immersion collection system. The immersioncollection system is one that optically couples the first lens ofoptical condenser subsystem 44 to the flow cell 28 by means of arefractive index matching layer, enabling collection of light over awide angle. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 5-31.

The condenser 44 is an optical lens subsystem with aberration correctionsufficient for diffraction limited imaging used in high resolutionmicroscopy. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 32-60.

The functions of other components shown in FIG. 1, i.e., a slit 46, afield lens 48, and a second slit 50, are described in U.S. Pat. No.5,631,165, at column 44, line 63 through column 45, line 15.Photomultiplier tubes 34 and 36 detect side scattering (light scatteredin a cone whose axis is approximately perpendicular to the incidentlaser beam), while photomultiplier tube 38 detects fluorescence (lightemitted from the cells at a different wavelength from that of theincident laser beam).

For most measurements, the only reagent used is a sheath diluent. Thesheath diluent used need be no different than that used on currenthematology analyzers, e.g., the CELL-DYN® 3000 series and the CELL-DYN®4000 series of hematology analyzers, although a different formulationcan also be employed if desired. The sheath diluent can include as areagent component a surfactant used to produce sphericity in the redblood cells in the sample.

In order to detect reticulocytes and nucleated red blood cells (NRBC), anucleic acid dye can be employed. Fluorescent nucleic acid dyes suitablefor use in the method described herein include, but are not limited to,a cell-permeable cyanine nucleic-acid stain that emits blue/violet lightsuch as SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44 and SYTO 45, (see,e.g., Wlodkowic (2008), Cytometry A 73, 496-507), both of which arecommercially available from Life Technologies, as well as othercell-permeable dyes with similar absorption and emission spectra as aSYTO dye, e.g., a dye that has an absorbance maximum in the range ofabout 425 to about 440 nm (e.g., in the range of about 428 nm to about435 nm) and an emission maximum in the range of about 450 nm to about465 nm (e.g., in the range of about 452 nm to 463 nm), although dyeshaving emission/absorption maxima outside of these ranges may beemployed in certain cases. Such dyes are collectively called “SYTO” dyesherein. Nucleic acid dyes that can be employed in the subject method mayabsorb light at the wavelength of the laser used, and emit light at awavelength that is different from that of the wavelength of the laser,thereby allowing fluorescence to be detected. In one embodiment, thenucleic-acid dye binds to both RNA and DNA in the sample. In analternative embodiment, two separate nucleic-acid dyes are employed, oneof them binding preferentially to RNA and the other bindingpreferentially to DNA. In a preferred embodiment, the one or morenucleic acid dye employed in the method displays fluorescenceenhancement upon binding, in order to reduce fluorescence background andimprove the signal-to-noise ratio.

With use of a laser having a wavelength in the range of from about 400nm to about 450 nm, red blood cells can be distinguished from whiteblood cells by a trigger value in forward scattering (IAS). A low IAStrigger value identifies platelets and red blood cells, thereby allowingthose cells to be counted. A high IAS trigger value identifies whiteblood cells, thereby allowing white blood cells to be counted and redblood cells and platelets to be automatically rejected, and permitting awhite blood cell differential. The low trigger value may in certaincases be 0.1 V. However, that value is dependent on factors such as therange of angles collected by the IAS detector, the efficiency of lightcollection, the quantum efficiency of the photodiode that is being usedas the IAS (intermediate angle scatter) detector, the gain of thephotodiode, the gain of any preamplifiers and amplifiers used, and, assuch, the trigger value can be in the range of 0.005V to 10V, e.g., 2.3V, depending on these factors.

The measurement process begins as the cell stream, having been dilutedwith the sheath diluent and being injected at such a rate thatmeasurement of red blood cells incurs in a satisfactorily low level ofcoincidences, passes through the flow cell 28 in a laminar flowingsample stream surrounded by a sheath solution. The illuminated volume isbounded in the two directions normal to the flow axis by thehydrodynamically focused cell stream, and in the dimension parallel tothe flow axis by the vertical beam waist of the laser beam, which isabout 17 micrometers. The flow rate of the diluted sample in the samplestream is about 0.5 microliters per second for the red blood cell andplatelet assay, and about 5 microliters per second for the white bloodcell assay.

FIG. 2 shows an example of the subject method. Referring to FIG. 2, theinitial stages of the sample preparation can be similar to those of theprior art, with the analogous steps of sample loading (1702), samplehomogenization (1704), and sample aspiration (1706). In comparison tothe prior art, the step of providing aliquots of the sample can beeliminated, because a single volume of the sample is used forprocessing. The three or more separate assays of the prior art can becombined into a single assay (1710), which yields those parameters thatin the prior art require an assay for red blood cell and plateletparameters, an assay for white blood cells, white blood celldifferential, and nucleated red blood cell parameters, an assay forquantification of reticulocyte parameters, and an assay forquantification of parameters related to hemoglobin. The volume of sampleis delivered to a single container (1714), and the diluent solution ismetered (1712) and delivered to the same container (1716). In oneembodiment, the ratio of diluent/sheath reagent to blood is about 100:1.The resulting mixture is homogenized (2318); the resulting mixture isthen incubated for a length of time dependent on the nature of thereagent (e.g., whether one or more nucleic-acid dyes are employed) andon the types of assays desired to be performed on the blood sample(e.g., whether a reticulocyte assay, an erythroblast assay, or bothassays are performed in addition to the standard CBC). The mixture ofthe sample and the reagent solution is then staged (1722) andimmediately passed through the flow cell, where the flow cytometermeasurements take place (1724). There are no separate sample mixtures tobe processed sequentially; therefore, the sample is directed to wasteand the flow cell rinsed (1726), thereby allowing another sample tofollow immediately. For the measurement of the white blood celldifferential, a rate of sample injection into the flow cell higher thanthe rate used for the count of red blood cells and platelets is used.

The signals from the flow cell measurements are processed and analyzedby the algorithm(s) (1728). The algorithms of the system candifferentiate each cell population based on the optical signalintensities recorded for each cell on each detector channel, and thelocation and pattern of each cluster of cells in cytograms constructedfrom combinations of two or more of the optical measurements, or inhistograms constructed from projections of the optical measurementsalong a chosen axis.

In particular embodiments, a universal clustering algorithm foranalyzing the data can be used. Such an algorithm can performsatisfactorily with any kind of data. Such a universal clusteringalgorithm is described below.

A universal clustering algorithm can create groups of similar events(clusters) in a high-dimensional space (larger than 2). The algorithmcan function with any reagent on any automated hematology analyzer. Thisis the first step of a data-driven algorithm. Algorithms of the priorart are knowledge-driven, i.e., what is being sought is known to someextent, and the data are categorized accordingly. Knowledge-drivenalgorithms can provide unusual results if the data are not as expected.Because in knowledge-driven algorithms assumptions are made about thedata, data sets that fall outside the set defined by those assumptionsmay not be analyzed correctly.

Data-driven algorithms can function with any data, even data thatcontain unexpected events. Such algorithms can always provide the mainclusters. Based on the output of this type of algorithm, the kind ofdata being observed can be determined, and then an appropriate algorithm(including knowledge-based algorithms) for the detailed analysis can beused. A clustering algorithm allows a coarse classification of data type(based on few, if any, broad assumptions) before a second algorithmexecutes a detailed classification based on more stringent assumptions.

In what follows, one embodiment of a clustering algorithm approach isdescribed. A k-means clustering routine is used to obtain an initial setof about 10 clusters. The number 10 is chosen to be higher than thenumber of meaningful clusters that are expected to be present in thedata sets to be analyzed. After the k-means routine returns, theclusters are merged, based on some measure of their distance. A fixeddistance threshold value can be used, and when the distance between twoclusters is below that value, those clusters can be merged. Mergingcontinues until there are no more pairs of clusters remaining that arecloser to each other than the cut-off value. Unlike the k-means routine,the clustering routine described herein returns a variable number ofclusters.

The distance measure described herein is a combination of clusterlocations and cluster spreads.

In certain cases, the data is clustered by the k-means method, whichaims to partition the points into k groups such that the sum of squaresfrom points to the assigned cluster centers is minimized. At theminimum, all cluster centers are at the mean of their Voronoi sets (theset of data points which are nearest to the cluster center).

The algorithm of Hartigan and Wong (Hartigan, J. A., and Wong, M. A.(1979), “Algorithm AS 136: A K-means clustering algorithm,” Journal ofthe Royal Statistical Society, Series C (Applied Statistics) 28,100-108) can be used by default. In certain cases a specific algorithmcan be employed, such as those described by MacQueen (MacQueen, J.(1967), “Some methods for classification and analysis of multivariateobservations,” in Proceedings of the Fifth Berkeley Symposium onMathematical Statistics and Probability, eds L. M. Le Cam & J. Neyman,1967 1, pp. 281-297. Berkeley, Calif.: University of California Press),Lloyd (Lloyd, S. P. (1957), “Least squares quantization in PCM,”Technical Note, Bell Laboratories. Published in 1982 in IEEETransactions on Information Theory 28, 128-137) and Forgy (Forgy, E. W.(1965), “Cluster analysis of multivariate data: efficiency vs.interpretability of classifications,” Biometrics 21, 768-769). TheHartigan-Wong algorithm can also be employed, but trying several randomstarts is often recommended. Except for the Lloyd-Forgy method, kclusters will always be returned if a number is specified. If an initialmatrix of centers is supplied, it is possible that no point will beclosest to one or more centers, which is currently an error for theHartigan-Wong method. The foregoing publications are incorporated hereinby reference.

The clustering algorithm used need not require any assumptions about thedata. The clustering algorithm used can return an adequate summary ofthe main components of the data observed. From the data observed,assumptions can be made about the kind of a sample being observed.Detailed algorithms that are tailored to the different samplesencountered can then be employed.

With a clustering approach as the first step in the classificationalgorithm, a very good rough classification of the sample can beobtained, and then an appropriate detailed algorithm can be run. Asecond advantage of clustering algorithms is that they can moreaccurately locate small populations, such as, for example, immaturegranulocytes.

Certain embodiments of the method described herein have advantages. Incertain cases, no lysing agent is required, thereby increasing theaccuracy of the count of white blood cells. Further, in certain cases,no lysing step is required, thereby increasing the throughput of theanalyzer. A strong hemoglobin signal allows the measurement of thehemoglobin concentration in red blood cells on a cell-by-cell basis asthey pass through the flow cell for enumeration. Furthermore, in certaincases, the method only requires a single dilution of the blood sample,thereby simplifying the fluidics of the hematology analyzer.

Certain embodiments of the apparatus and method described herein providea significant reduction in the complexity of the analyzer, in the numberof separate processing steps required for a standard complete bloodcount, in the number and amount of reagents used to obtain a completeblood count, in the cost of its manufacture, in the risk of failureduring operation, and in the cost of maintenance and service.Furthermore, the apparatus and method described herein can eliminate theneed to lyse red blood cells during the white blood cell assay, therebyeliminating the interference with the white blood cell count ordifferential assay from lyse-resistant red blood cells (including, e.g.,sickle cells, target cells, and erythroblasts from neonates).

In one embodiment, the red blood cell lysis incubation step is notperformed and, in certain embodiments, the multiple assays of aconventional method are combined into a single assay. For example, eachof the two or more delivery subsystems of the lysing procedure of theprior art that is eliminated by adoption of the single dilutionprocedure of the present invention may include the following components:(a) a precision metering syringe; (b) a syringe assembly; (c) a syringestepper motor; (d) a stepper motor driver board; (e) several lengths ofnoncompliant tubing; (f) several pinch valves; (g) the correspondingpilot valves that operate the pinch valves, or alternatively thesolenoids operating the pinch valves; (h) the electronic boardcomponents driving the pilot valves or the solenoids; (i) a containerused to mix one aliquot of sample with the metered quantity of reagent;(j) a motor used to mix the sample aliquot with the reagent solution;(k) the mixer motor driver board; (l) the firmware for controlling thestepper motor, the mixer motor, and the several pilot valves orsolenoids; (m) the power source(s) for the stepper motor, the mixermotor, and the pilot valves or solenoids; (n) the fans for removing theheat from the flow panel due to operation of the pilot valves orsolenoids. Taking as example the CELL-DYN® Sapphire® hematologyanalyzer, where three reagent delivery subsystems supporting flowcytometry measurements are currently in use (that for the red bloodcell/platelet assay; that for the white blood cell, white blood celldifferential, and nucleated red blood cell assay; and that for theoptional reticulocyte assay), adoption of the apparatus and methoddescribed herein would reduce these to a single reagent deliverysubsystem. Subsystems supporting impedance measurements (for cell volumedetermination) or colorimetric measurements (for bulk hemoglobindetermination) need not be affected. However, these subsystems, too,could optionally be eliminated altogether for additional benefits insimplicity, reliability, and cost, because the apparatus and method usedfor a lysis-free single-dilution approach could provide all of thereportable parameters (including mean cell volume and overall hemoglobinconcentration) that are required of a commercial hematology analyzer.

The reagents used in the analyzer can be reduced relative to the set inthe prior art (which includes a lysing agent for use in the white bloodcell assay, an optional nucleic acid dye added to the lysing agent foruse in the concurrent nucleated red blood cell assay, a diluent solutioncontaining optional sphering reagent for the red blood cell/plateletassay, a reagent solution used for the reticulocyte assay, which reagentsolution includes a nucleic acid dye, and a strong lysing agent used forhemoglobin quantification) to a single reagent solution, which comprisesa diluent, typically a saline diluent. The single reagent solutionpreferably comprises an isovolumetric sphering reagent, such as, e.g., asurfactant, acting on the membrane of the red blood cells to confer uponthem an approximately spherical form while leaving their volumesubstantially unchanged, in order to prevent orientation-dependent lightscattering results from otherwise essentially equivalent cells. Thesingle reagent solution optionally comprises one or two nucleic aciddyes for reticulocyte and nucleated red blood cell analysis. At leastone of the nucleic acid dyes should be capable of staining RNA, and atleast one of the nucleic acid dyes should be capable of staining DNA.Alternatively, the at least one nucleic acid can be capable of stainingboth RNA and DNA. Another optional ingredient of the reagent solutionfor use in the method described herein is a selective permeabilizingagent. Only one dilution ratio is used. The cell counting andidentification algorithms are combined from a set dedicated to each ofthe currently employed assays to a single set to be applied to thesingle assay being performed. Furthermore, the algorithms employ similardata (signals) to those that are currently employed. The precision ofresults can be automatically maintained by design. The coincidencelevels can be maintained by design.

The method described above can be employed to measure the number ofindividual red blood cells, the volume of individual red blood cells,and the amount of hemoglobin in individual red blood cells of a sample,thereby permitting the analysis of further characteristics of thepopulation. In one embodiment, the method can further comprisecalculating the proportion (which can, for example, be expressed as apercentage, fraction or another number), of red blood cells having adefined characteristic. For example, the method can be employed tocalculate the proportion of red blood cells having a volume above and/orbelow a defined volume (e.g., the percentage of cells larger than 120fL, i.e., the percentage of “macrocytic” red blood cells; or thepercentage of cells smaller than 60 fL, i.e., the percentage of“microcytic” red blood cells). In another embodiment, the method can beemployed to calculate the proportion of red blood cells having ahemoglobin concentration above and/or below a defined volume (e.g., thepercentage of red blood cells having a cellular hemoglobin concentrationof less then 28 g/dL, i.e., the percentage of “hypochromic” red bloodcells; or the percentage of red blood cells having a cellular hemoglobinconcentration of greater than 41 g/dL, i.e., the percentage of“hyperchromic” red blood cells). Likewise, the volumes and/or hemoglobinconcentrations of individual red blood cells of a population can bestatistically analyzed to identify other statistical measures thatdescribe, for example, the shape of the distribution or variation of thevolume or hemoglobin concentration of individual RBCs within thepopulation. In one exemplary embodiment, the width of the distributionof hemoglobin concentration in the population of red blood cells iscalculated.

In a further embodiment, the method can further involve identifyingreticulocytes in the sample. Reticulocytes are a subset of red bloodcells that are distinguishable from other red blood cells by, e.g.,fluorescence. The method can also be used to further analyze thereticulocytes in a sample by, for example, calculating the mean amountof hemoglobin in the reticulocytes, the mean concentration of hemoglobinin the reticulocytes, or the mean volume of the reticulocytes. Themethod can further be employed to present the reticulocyte population incomparison to the mature red blood cell population on appropriatecytograms, e.g., on a cytogram displaying the volume vs. hemoglobinconcentration of individual reticulocytes and red blood cells in thesample.

The hematology analyzer described above can be employed, for example, toinvestigate red blood cell disorders or anemias, and to make treatmentdecisions, if necessary. Examples of anemia include iron deficiencyanemia, anemia of chronic disorder, and megaloblastic anemia caused byvitamin B₁₂ or folic acid. For example, administration of ironsupplement is extremely effective as a treatment for iron deficiencyanemia, but not for anemia of chronic disorder. The cause of the anemiais therefore important to the treatment of the anemia.

Iron deficiency is the most prevalent single deficiency state on aworldwide basis. It is important economically because it diminishes thecapability of affected individuals to perform physical labor, and itdiminishes both growth and learning in children.

Absolute iron deficiency, with anemia or without anemia, and functionaliron deficiency are high-frequency clinical conditions, and patientswith these conditions have iron-deficient erythropoiesis. Absolute irondeficiency is defined as a decrease in total body iron content.Iron-deficiency anemia occurs when iron deficiency is sufficientlysevere to diminish erythropoiesis and cause the development of anemia.Functional iron deficiency describes a state where the total ironcontent of the body is normal or even elevated, but the iron is “lockedaway” and unavailable for the production of red blood cells. Thiscondition is observed mainly in patients with chronic renal failure whoare on hemodialysis, and in patients with chronic inflammation orchronic infections.

Iron status can be measured using hematological and biochemical indices.Each parameter of iron status reflects changes in different forms ofbody iron storage and is affected at different levels of iron depletion.Specific iron measurements include hemoglobin, mean cell volume,hematocrit, erythrocyte protoporphyrin, plasma iron, transferrin,transferrin saturation levels, serum ferritin, soluble transferrinreceptors, and red-cell distribution width.

Hemoglobin has been used longer than any other iron status parameter. Itprovides a quantitative measure of the severity of iron deficiency onceanemia has developed. Hemoglobin determination is a convenient andsimple screening method and is especially useful when the prevalence ofiron deficiency is high, as in pregnancy or infancy. The limitations ofusing hemoglobin as a measure of iron status are its lack of specificity(since factors such as vitamin B₁₂ or folate deficiency, geneticdisorders and chronic infections can limit erythropoiesis) and itsrelative insensitivity due to the marked overlap in values betweennormal and iron-deficient populations. To identify iron-deficiencyanemia, hemoglobin is measured together with more selective measurementsof iron status.

A reduction in mean cell volume occurs when iron deficiency becomessevere, at about the same time that anemia starts to develop. It is afairly specific indicator of iron deficiency once thalassemia and theanemia of chronic disease have been excluded. A cut-off value of 80 flis accepted as the lower limit of the normal range in adults. Thered-cell distribution width (RDW) has been used recently in combinationwith other parameters for the classification of anemias. RDW reflectsthe variation in the size of the red cells and can be used to detectsubtle degrees of anisocytosis.

The most commonly used iron status parameters at present are transferrinsaturation (TSAT) and serum ferritin. However, both are indirectmeasures of iron status. Transferrin is a transport protein thatcontains two iron binding sites by which it transports iron from storagesites to erythroid precursors. TSAT (i.e., the percentage of totalbinding sites that are occupied by iron) is a measure of iron that isavailable for erythropoiesis. TSAT is calculated by dividing the serumiron by the total iron binding capacity, a measurement of circulatingtransferrin, and multiplying by 100. Ferritin is a storage protein thatis contained primarily within the reticuloendothelial system, with someamounts released in the serum. Under conditions of iron excess, ferritinproduction increases to offset the increase in plasma iron. The level offerritin in the serum, therefore, reflects the amount of iron instorage.

Reticulocytes are immature red blood cells with a maturation time ofonly 1 to 2 days before turning into mature red blood cells. Whenreticulocytes are first released from the bone marrow, measurement oftheir hemoglobin content can provide the amount of iron immediatelyavailable for erythropoiesis. A less-than-normal hemoglobin content inthese reticulocytes is an indication of inadequate iron supply relativeto demand. The amount of hemoglobin in these reticulocytes alsocorresponds to the amount of hemoglobin in mature red blood cells. Thehemoglobin content of reticulocytes has been evaluated recently innumerous studies as a test for absolute iron deficiency and functionaliron deficiency and has been found to be highly sensitive and specific.However, exact threshold values have not been established, because thethreshold values vary depending on the laboratory and instrument used.

Erythropoietin is effective in stimulating production of red bloodcells, but without an adequate iron supply to bind to hemoglobin, thered blood cells will be hypochromic, i.e., low in hemoglobin content.Thus, in states of iron deficiency, a significant percentage of redblood cells leaving the bone marrow will have low hemoglobin content. Bymeasuring the percentage of red blood cells with hemoglobin content lessthan 28 g/dL, iron deficiency can be detected. A percentage ofhypochromic cells greater than 10% has been correlated with irondeficiency, and hence has been used as a diagnostic criterion fordetection of iron deficiency.

In addition to providing a white blood cell count, the above-describedapparatus and method can provide a white blood cell differential thatprovides the percentage of each type of white blood cell (WBC) in ablood sample, thereby revealing if there are certain pathologies likelyaffecting the white blood cells in the sample. Neutrophils can increasein response to bacterial infection or inflammatory disease. Severeelevations in neutrophils may be caused by various bone marrowdisorders, such as chronic myelogenous leukemia. Decreased neutrophillevels may be the result of severe infection or other conditions, suchas responses to various medications, particularly chemotherapy.Eosinophils can increase in response to allergic disorders, inflammationof the skin, and parasitic infections. They can also increase inresponse to some infections or to various bone marrow disorders.Decreased levels of eosinophils can occur as a result of infection.Basophils can increase in cases of leukemia, chronic inflammation, thepresence of a hypersensitivity reaction to food, or radiation therapy.Lymphocytes can increase in cases of viral infection, leukemia, cancerof the bone marrow, or radiation therapy. Decreased lymphocyte levelscan indicate diseases that affect the immune system, such as lupus, andthe later stages of HIV infection. Monocyte levels can increase inresponse to infection of all kinds as well as to inflammatory disorders.Monocyte counts are also increased in certain malignant disorders,including leukemia. Decreased monocyte levels can indicate bone marrowinjury or failure and some forms of leukemia. Since percentages might bemisleading in some patients, absolute values of the various types ofWBCs can also be reported, such as the absolute neutrophil count (ANC).Absolute values are calculated by multiplying the number of WBCs by thepercentage of each type of white cell and can aid in diagnosing illnessand monitoring therapy.

In one embodiment, a physical memory containing instructions (i.e.“programming”) for performing the method described herein is provided.In some embodiments, the memory can comprise a physicalcomputer-readable medium comprising: i. programming to differentiate andcount white blood cells in a sample using multiple in-flow opticalmeasurements obtained from a first portion of the sample that has beenintroduced into a flow cell at a high rate and ii. programming todifferentiate and count red blood cells and platelets in the sampleusing multiple in-flow optical measurements obtained from a secondportion of the sample that has been introduced into a flow cell at a lowrate. In one embodiment, data from the hematology analyzer is collected,and programming containing an algorithm for the calculation is executed,using the multiple in-flow optical measurements as inputs.

The programming can be provided in a physical storage or transmissionmedium. A computer receiving the instructions can then execute thealgorithm and/or process data obtained from the subject method. Examplesof storage media that are computer-readable include floppy disks,magnetic tape, CD-ROM, a hard disk drive, a ROM or integrated circuit, amagneto-optical disk, or a computer readable card such as a PCMCIA cardand the like, whether or not such devices are internal or external tothe computer. A file containing information can be “stored” on computerreadable medium, where “storing” means recording information such thatit is accessible and retrievable at a later date by a computer on alocal or remote network.

The method described herein can be automatically executed each time asample is run, or each can be executed on a sample in response todifferent test selections by an operator.

The following non-limiting examples illustrate the method of thisinvention. In the following examples, the parameters that are measuredare described as follows:

-   -   (1) size, or 0° channel: this detection channel measures two        different quantities, depending on the analyzer platform:        -   (a) ALL: extinction, i.e., light lost from the main beam            (e.g., on the CELL-DYN® Sapphire®); or        -   (b) SAS: light scattered at angles ranging from about 1° to            about 3° with respect to the laser beam propagation axis            (e.g., on the CELL-DYN® Ruby®)    -   (2) IAS, complexity, 7° or 10° channel: light scattered at        angles ranging from about 3° to about 10° with respect to the        laser beam propagation axis.    -   (3) PSS, lobularity, or 90° channel: light scattered        orthogonally to the laser beam which maintains vertical        polarization.    -   (4) DSS, granularity, or 90° depolarized channel: light        scattered orthogonally to the laser beam, which by interaction        with cellular subcomponents has acquired horizontal        polarization.

Example 1

This example illustrates separation of red blood cells, white bloodcells, and platelets in a sample of unlysed whole blood carried out on aprototype analyzer that uses a laser having a wavelength of 405 nm. Theresults are shown in FIG. 3. It can be seen that there is clearseparation between platelets, red blood cells, and white blood cells.The number of white blood cells is too low to see a white blood celldifferential. In order to collect more white blood cell events, an IAStrigger value corresponding to about 8000 on the IAS is required.

Example 2

This example illustrates a white blood cell differential carried out ona prototype analyzer that uses a laser having a wavelength of 405 nm,wherein the method does not employ a lysing agent. The results are shownin FIG. 4, where monocytes are designated by the letter “M”, lymphocytesare designated by the letter “L”, neutrophils are designated by theletter “N”, and eosinophils are designated by the letter “E.” Theclassification is achieved without a clustering algorithm.

Example 3

This example illustrates that issues encountered with lysis-resistantred blood cells interfering with the white blood cell count by theCELL-DYN® Ruby® hematology analyzer and the CELL-DYN® Sapphire®hematology analyzer are significantly improved with a laser having awavelength of 405 nm. CELL-DYN® Ruby® hematology analyzers employ awavelength of 633 nm. CELL-DYN® Sapphire® hematology analyzers employ awavelength of 488 nm. The same blood sample was run on three differentanalyzers using four separate assays; the results are described below,with reference to FIGS. 5-8.

FIGS. 5A, 5B, 5C, and 5D show that the results generated by the analyzerwould indicate that the sample has a lymphocyte percentage of 95%, asmeasured with a CELL-DYN® Sapphire® hematology analyzer employing alaser having a wavelength outside the range of 400 nm to 450 nm and alysing agent of low lytic strength. FIGS. 6A, 6B, 6C, and 6D show thatthe results generated by the analyzer would indicate that the sample hasa lymphocyte percentage of 48%, as measured with a CELL-DYN® Sapphire®hematology analyzer employing a laser having a wavelength outside therange of 400 nm to 450 nm and a lysing agent of high lytic strength.FIGS. 7A, 7B, 7C, and 7D show that the results generated by the analyzerwould indicate that the sample has a lymphocyte percentage of 55%, asmeasured with a CELL-DYN® Ruby® hematology analyzer employing a laserhaving a wavelength outside the range of 400 nm to 450 nm and a lysingagent of intermediate strength.

FIG. 8 indicates that the sample has a lymphocyte percentage of 12%, asmeasured with a prototype analyzer equipped with a 405 nm laser, but notemploying a lysing agent and using the single dilution techniquedescribed herein. Here the lymphocyte percentage is measured to be 12%.The lymphocytes are designated by the letter “L.” The reference methodused to establish truth for this particular sample is a microscope slidereview by a trained operator, which provided for this sample alymphocyte percentage of 10%. The use of the 405 nm laser and the singledilution technique described herein provided results that were closestto those of the reference method, whereas both the CELL-DYN® Sapphire®hematology analyzer (for both assays with low lytic strength and highlytic strength) and the CELL-DYN® Ruby® hematology analyzer providedresults that were significantly discrepant due to interference fromlysis-resistant red blood cells. The data show that the hematologyanalyzer using a 405 nm laser is superior for reducing or eliminatinginterference from lysis-resistant red blood cells in the measurement ofwhite blood cell count and in the white blood cell differential.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

What is claimed is:
 1. A system for counting white blood cells, redblood cells, and platelets in a blood sample, the system comprising: ahematology analyzer, wherein the hematology analyzer comprises: a) aflow cell; b) a light source; c) a plurality of detectors configured todetect light scattering by cells and platelets of the blood sample; andd) a data analysis workstation comprising a processor, wherein theprocessor comprises a non-transient, computer-readable medium programmedwith instructions that, when executed by the processor, cause thehematology analyzer to: i. flow a first portion of the blood sample intothe flow cell of the hematology analyzer at a first flow rate and directlight to the flow cell to generate first flow rate optical data usingthe plurality of detectors, wherein the first flow rate optical datacomprise light scattering by cells and platelets in the first portion ofthe blood sample; ii. flow a second portion of the same blood sampleinto the flow cell, directly following the first portion of the bloodsample, at a second flow rate, the second flow rate being lower than thefirst flow rate, and direct light to the flow cell to generate secondflow rate optical data using the plurality of detectors, wherein thesecond flow rate optical data comprise light scattering by cells andplatelets in the second portion of the blood sample; and iii.simultaneously count: white blood cells in the blood sample using thefirst flow rate optical data; and red blood cells and platelets in theblood sample using the second flow rate optical data.
 2. The system ofclaim 1, wherein the light source is selected from the group consistingof: a lamp, a light-emitting diode (LED), and a laser.
 3. The system ofclaim 2, wherein the light source is a laser.
 4. The system of claim 1,wherein the system is configured to analyze a blood sample that is nottreated to lyse the red blood cells.
 5. The system of claim 4, whereinthe light directed to the flow cell has a wavelength ranging from 400 nmto 450 nm.
 6. The system of claim 4, wherein the light directed to theflow cell has a wavelength ranging from 400 nm to 430 nm.
 7. The systemof claim 1, wherein the plurality of detectors is selected from thegroup consisting of: an axial light loss (ALL) detector, a small anglescattering (SAS) detector, an intermediate angle scattering (IAS)detector, a polarized side scattering (PSS) detector, a depolarized sidescattering (DSS) detector, a fluorescence detector, and any combinationthereof.
 8. The system of claim 7, wherein the first flow rate opticaldata comprises an amplitude of a signal generated by the white bloodcells that is above an IAS trigger threshold.
 9. The system of claim 1,wherein the plurality of detectors comprises a detector for obtaininglight extinction measurements at an angle of from 0° to 1°.
 10. Thesystem of claim 1, wherein the one or more detectors comprises adetector for obtaining light scattering measurements at an angle of from3° to 10°.
 11. The system of claim 1, wherein the first flow rate is tenor more times greater than the second flow rate.
 12. The system of claim1, wherein the first flow rate is such that from 0 to 50,000 white bloodcells are introduced into the flow cell per second.
 13. The system ofclaim 1, wherein the second flow rate is such that from 50,000 to300,000 red blood cells are introduced into the flow cell per second.14. The system of claim 1, wherein the non-transient, computer-readablemedium further comprises instructions for counting white blood cells inthe blood sample using the first flow rate optical data.
 15. The systemof claim 14, wherein the non-transient, computer-readable medium furthercomprises instructions for differentiating the white blood cells usingthe first flow rate optical data and at least one algorithm todifferentiate and count different types of white blood cells.
 16. Thesystem of claim 1, wherein the non-transient, computer-readable mediumfurther comprises instructions for counting red blood cells andplatelets in the blood sample using the second flow rate optical data.17. The system of claim 16, wherein the non-transient, computer-readablemedium further comprises instructions for differentiating the red bloodcells and platelets in the blood sample using the second flow rateoptical data.
 18. The system of claim 1, wherein the non-transient,computer-readable medium further comprises instructions for:differentiating and counting a plurality of white blood cells in thesample of whole blood using the first flow rate optical data and atleast one algorithm to differentiate and count different types of whiteblood cells; and differentiating and counting a plurality of red bloodcells and platelets in the sample of whole blood using the second flowrate optical data.
 19. The system of claim 1, wherein the non-transient,computer-readable medium further comprises instructions for identifyinga red blood cell disorder selected from the group consisting of: irondeficiency anemia, anemia of chronic disorder, and megaloblastic anemia.